Specific substitutions at amino acid 256 of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase mediate resistance to thapsigargin in thapsigargin-resistant hamster cells.
High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T --> C change at nucleotide 766 introduces a Phe256 --> Leu alteration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 microM, a T --> C change at nucleotide 767 introduces a Phe256 --> Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4-5-fold resistance to TG inhibition of Ca2+ transport function occurs upon the introduction of either the Phe256 --> Leu or the Phe256 --> Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.
Yu
M
M
Division of Oncology, Department of Medicine, Greenebaum Cancer Center, Baltimore, Maryland 21201, USA.
Zhong
L
L
Rishi
A K
AK
Khadeer
M
M
Inesi
G
G
Hussain
A
A
Zhang
L
L
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology; D008647:Mesocricetus; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology; D019284:Thapsigargin / Q000494:pharmacology*
D000596:Amino Acids; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases